Deubiquitinase USP7 contributes to the pathogenicity of spinal and bulbar muscular atrophy.

Polyglutamine (polyQ) diseases are devastating, slowly progressing neurodegenerative conditions caused by expansion of polyQ-encoding CAG repeats within the coding regions of distinct, unrelated genes. In spinal and bulbar muscular atrophy (SBMA), polyQ expansion within the androgen receptor (AR) causes progressive neuromuscular toxicity, the molecular basis of which is unclear. Using quantitative proteomics, we identified changes in the AR interactome caused by polyQ expansion. We found that the deubiquitinase USP7 preferentially interacts with polyQ-expanded AR and that lowering USP7 levels reduced mutant AR aggregation and cytotoxicity in cell models of SBMA. Moreover, USP7 knockdown suppressed disease phenotypes in SBMA and spinocerebellar ataxia type 3 (SCA3) fly models, and monoallelic knockout of Usp7 ameliorated several motor deficiencies in transgenic SBMA mice. USP7 overexpression resulted in reduced AR ubiquitination, indicating the direct action of USP7 on AR. Using quantitative proteomics, we identified the ubiquitinated lysine residues on mutant AR that are regulated by USP7. Finally, we found that USP7 also differentially interacts with mutant Huntingtin (HTT) protein in striatum and frontal cortex of a knockin mouse model of Huntington's disease. Taken together, our findings reveal a critical role for USP7 in the pathophysiology of SBMA and suggest a similar role in SCA3 and Huntington's disease.

Targeting alpha synuclein and amyloid beta by a multifunctional, brain-penetrant dopamine D2/D3 agonist D-520: Potential therapeutic application in Parkinson's disease with dementia.

A significant number of people with Parkinson's disease (PD) develop dementia in addition to cognitive dysfunction and are diagnosed as PD with dementia (PDD). This is characterized by cortical and limbic alpha synuclein (α-syn) accumulation, and high levels of diffuse amyloid beta (Aß) plaques in the striatum and neocortical areas. In this regard, we evaluated the effect of a brain-penetrant, novel multifunctional dopamine D2/D3 agonist, D-520 on the inhibition of Aß aggregation and disintegration of α-syn and Aß aggregates in vitro using purified proteins and in a cell culture model that produces intracellular Aß-induced toxicity. We further evaluated the effect of D-520 in a Drosophila model of Aß1-42 toxicity. We report that D-520 inhibits the formation of Aß aggregates in vitro and promotes the disaggregation of both α-syn and Aß aggregates. Finally, in an in vivo Drosophila model of Aß1-42 dependent toxicity, D-520 exhibited efficacy by rescuing fly eyes from retinal degeneration caused by Aß toxicity. Our data indicate the potential therapeutic applicability of D-520 in addressing motor dysfunction and neuroprotection in PD and PDD, as well as attenuating dementia in people with PDD.

Expression and Regulation of Deubiquitinase-Resistant, Unanchored Ubiquitin Chains in Drosophila.

The modifier protein, ubiquitin (Ub) regulates various cellular pathways by controlling the fate of substrates to which it is conjugated. Ub moieties are also conjugated to each other, forming chains of various topologies. In cells, poly-Ub is attached to proteins and also exists in unanchored form. Accumulation of unanchored poly-Ub is thought to be harmful and quickly dispersed through dismantling by deubiquitinases (DUBs). We wondered whether disassembly by DUBs is necessary to control unanchored Ub chains in vivo. We generated Drosophila melanogaster lines that express Ub chains non-cleavable into mono-Ub by DUBs. These chains are rapidly modified with different linkages and represent various types of unanchored species. We found that unanchored poly-Ub is not devastating in Drosophila, under normal conditions or during stress. The DUB-resistant, free Ub chains are degraded by the proteasome, at least in part through the assistance of VCP and its cofactor, p47. Also, unanchored poly-Ub that cannot be cleaved by DUBs can be conjugated en bloc, in vivo. Our results indicate that unanchored poly-Ub species need not be intrinsically toxic; they can be controlled independently of DUB-based disassembly by being degraded, or through conjugation onto other proteins.

Androgen receptor polyglutamine expansion drives age-dependent quality control defects and muscle dysfunction.

Skeletal muscle has emerged as a critical, disease-relevant target tissue in spinal and bulbar muscular atrophy, a degenerative disorder of the neuromuscular system caused by a CAG/polyglutamine (polyQ) expansion in the androgen receptor (AR) gene. Here, we used RNA-sequencing (RNA-Seq) to identify pathways that are disrupted in diseased muscle using AR113Q knockin mice. This analysis unexpectedly identified substantially diminished expression of numerous ubiquitin/proteasome pathway genes in AR113Q muscle, encoding approximately 30% of proteasome subunits and 20% of E2 ubiquitin conjugases. These changes were age, hormone, and glutamine length dependent and arose due to a toxic gain of function conferred by the mutation. Moreover, altered gene expression was associated with decreased levels of the proteasome transcription factor NRF1 and its activator DDI2 and resulted in diminished proteasome activity. Ubiquitinated ADRM1 was detected in AR113Q muscle, indicating the occurrence of stalled proteasomes in mutant mice. Finally, diminished expression of Drosophila orthologues of NRF1 or ADRM1 promoted the accumulation of polyQ AR protein and increased toxicity. Collectively, these data indicate that AR113Q muscle develops progressive proteasome dysfunction that leads to the impairment of quality control and the accumulation of polyQ AR protein, key features that contribute to the age-dependent onset and progression of this disorder.